Edible fungal protein from non-toxic penicillium

ABSTRACT

Penicillin free edible protein-containing substance suitable for human consumption is produced by incubating and proliferating, under aerobic conditions, a non-toxic and non-penicillin producing strain of Penicillium notatum or Penicillium chrysogenum or a variant or mutant thereof in a culture medium containing essential growth-promoting nutrient substances, of which carbon in the form of assimilable carbohydrate constitutes the limiting substrate in proliferation, and separating from the assimilable carbohydrate exhausted medium the proliferated organism which is penicillin free and which constitutes the edible protein-containing substance suitable for human consumption possessing a high net protein utilization value of at least 70 based on alpha-amino nitrogen.

United States Patent Spicer et al.

EDIBLE FUNGAL PROTEIN FROM NON-TOXIC PENICILLIUM Inventors: Arnold Spicer, Iver Heath; Gerald L. Solomons, High Wycombe, both of England Assignee: Ranks Hovis McDougalI Limited,

London, England Notice: The portion of the term of this patent subsequent to Feb. 11, 1992, has been disclaimed.

Filed: June 24, 1974 Appl. No.: 482,134

Related US. Application Data Division of Ser. No. 333,788, Feb. 20, 1973, Pat. No. 3,865,951, which is a continuation-in-panof Ser. No. 116,684, Feb. 18, 1971, abandoned.

[56] References Cited UNITED STATES PATENTS 3,151,038 9/1964 Gray 195/81 x Primary Examiner-James R. Hoffman Attorney, Agent, or Firm Stevens, Davis, Miller & Mosher ABSTRACT Penicillin free edible protein-containing substance suitable for human consumption is produced by incubating and proliferating, under aerobic conditions, a non-toxic and non-penicillin producing strain of Peni cillium notatum or Penicillium chrysogenum or a variant or mutant thereof in a culture medium containing essential growth'promoting nutrient substances, of which carbon in the form of assimilable carbohydrate constitutes the limiting substrate in proliferation, and separating from the assimilable carbohydrate exhausted medium the proliferated organism which is penicillin free and which constitutes the edible protein-containing substance suitable for human consumption possessing a high net protein utilization value of at least 70 based on alpha-amino nitrogen.

1 Claim, No Drawings Character of EDIBLE FUNGAL PROTEIN FROM NON-TOXIC PENICILLIUM This is a division of application Ser. No. 333,788, filed, Feb. 20, 1973 and now U.S. Pat. No. 3,865,951 which is a continuation of application Ser. No. 116,684, filed Feb. 18, 1971, now abandoned.

The present invention relates to a process for the production of edible protein-containing substances and has particular reference to the production of fungal protein by microbial action.

British Application No. 53312/66, now British Pat. No. 1210356, relates to a process for the production of an edible protein-containing substance which comprises incubating and proliferating, under aerobic con ditions, an organism which is a nontoxic strain of a microfungus of the class Fungi lmperfecti, in a culture medium containingessential growth-promoting nutrient substances, of which carbon in the form of assimila blecarbohydrate constitutes the limiting substrate in proliferation, and separating from a assimilable carbohydrate exhausted medium the proliferated organism which constitutes the edible protein-containing substance.

It is an object of the present invention to provide fungal mycelium as an unsupplemented protein source which possesses a high net protein utilisation value on rat assays of at least 70. based on the a-amino nitrogen.

According to the present invention there is provided a process for the production of an edible proteincontaining substance which comprises incubating and proliferating, under aerobic conditions, a non-toxic strain of Penicillium notatum or Penicillium chrysoge num or a variant or mutant thereof, in a culture medium containing essential growthpromoting nutrient substances, of which carbon in the form of assimilable carbohydrate constitutes the limiting substrate in proliferation, and separating from the assimilable carbohydrate exhausted medium the proliferated organism which constitutes the edible protein-containing substance.

The separated proliferated organism which constitute the edible protein-containing substance may be in- 'monwealth Mycological lnstitute, Kew, and assigned the number l.M.l.138291.

It has the following morphological characteristics Malt Extract Czapek'Dox (modified) Medium Agar (Oxoid) Agar (Oxoid) Growth conditions days C 10 days 25C Colony Characteristics Rate of growth 5.0-5.5 cm 4.5-5.0 cm diameter diameter in in 10 days 10 days Close textured, Close textured,

brown, no pigment abundant pigment visible in medium diffusing into medium Cm very similar on both media Penicilli: Biverticillate asymmetrical. Length (branch to phialides) l635p.

At colony margin arising from basal felt. smooth-walled. ZOO-300p. approx. Width 2-4p.

One or two branches usually, arising at same point on main stem. asymmetric. Length 9-20u. width 2-441.

Groups of 25, usually 3. Compact whorl. hot divergent, length 3l0p.. width 2-4'1.

Groups of 4-6. typical bottle-shaped cells. Lengths 4.5-6.0p.. width 2.0-3.0;1. Forming tangled chains up to p. in length. usually 8011.. Subglobose. 3-3.5p. X 2.53}L

smooth walled. yellowish green in mass Conidiophore:

Branches:

Metulae:

Phialides:

Conidia:

The penicilli are intermediate between P. chrysogenum and notatum in complexity with slightly elliptical spores like P. chrysogenum.

Our British Application No. 8978/70 also describes specifically two variants of our strain of Penicillium notatum-chrysogenum C1 namely [.113 and 1.195 deposited with the Commonwealth Mycological Institute and assigned the numbers l.M.l.l42385 and [.M- 1142386 respectively and two mutants of said strain Cl namely 1.64 and 1.156 likewise deposited with the Commonwealth Mycological Institute and assigned the numbers I.M.l.142383 and 142384 respectively.

I. 113 (l.M.l 142,385) and l. (1.M.l. 142,386) having the following morphological characteristics:

Medium Malt Extract Czapek-Dox (modified Agar (Oxoid) Agar (Oxoid) Growth conditions 10 days at 25C 10 days at 25C Colony characteristics Rate of growth 5.06.0 cm 45-50 cm in 10 days in 10 days Character of Floccose at first. Floccose particularly growth tending to become in center. Four radial velvety. Light furrows. radial furrows.

Margin Entire Entire Amount of sporulation Sparse Sparse Color White to pale White to pale lemon green Exudate Absent Absent Odor Very little Very little Reverse Yellow-brown no Bright yellow with pigment in medium abundant pigment in medium Conidial stage Similar on both media Penieilli: Biverticillate asymmetric. Length Conidiophore:

(branch to phialide) 20-50p. Smooth walled. approx. 200p. Width 2-4u -Continued 1. l 13 Medium Malt Extract Czapek-Dox (modified Agar (Oxoid) Agar (Oxoid) Branches: Usually single. occasionally two arising at same point on conidiophore. Length 20-40p. occasionally vestigial. Width 3-4p. Metulae: Usually two, occasionally three per branch. Length -l0y., usually p. Width Z-Sp. as some are swollen in center. Phialides: Oneor two per metula. typical bottle shape. Length 4-8 Width 2-3p. Conidia: Short chains up to 60p. in length usually 25-30;. Subglobose 2.5-3.5;1. X 2.0 3.0a smooth walled l. 195 Medium Malt Extract Czapek Dox (modified) Agar (Oxoid) Agar (Oxoid) Growth conditions l0 days at 25C 10 days at 25C Colony Characteristics Rate of growth 4.5-5.0 cm 3.5-4.0 cm in 10 days in 10 days Character of Floccose center As on M.E.A.

phialides) -50y. Smooth walled approx. 2-300u arising from fiaerial hyphae. Width 2-3y.

Single arising from several points on conidiophore. Length 5-40u. often vestigial 51.1. Width 2-3p.

,Qne per branch. occasionally two.

Length 5-l0p. (usually 10;). Width 3-4p. often swollen appearance One per metula. rather restricted in comparison with typical shape. Length 4-8;; Width 2p.

Very short chains, one to three spores, occasionally five. Subglobose 3.0-3.5 2:5-3.0p. smooth walled.

Conidiopho 'rez" Branches:

Metulae:

Phialides:

Conidia:

The mutant l. 64 (I.M.I. 142,383) has a morphology "identical to its parent CI. The mutant I. 156 has a morphology very similar to I. 113 except the colony is yellow-brown to cream in color in contrast to the pale green color of l. 113.

The-temperature of incubation is in general between and 35C., preferably around C.

Inoculationresulting in commencement of the process is best carried out by a pregerminated seed stage comprising for example from 2 to 10 percent of inoculum, usually in the range 5 to l0 percent.

The pH of the substrate medium during incubation is preferably kept within a suitable range supporting maximum growth, for example, between 4 to 7.

The period of growth in batch culture is usually found to range from 20 to 48 hours. In certain instances for example when the carbohydrate is in the form of lactose the time may be extended to 72 hours. In both batch and continuous processes aeration and agitation should be carried out to provide a sufficient level of dissolved oxygen to overcome oxygen deficiency which is a limiting growth factor.

As will be well understood by those skilled in the art sufficient quantities of essential growth nutrients such as nitrogen, sulphur, phosphorus and other trace elements are maintained in the substrate medium so that growth of the substance is limited only by the carbohydrate available to the fungus.

In addition to the nutrients stated above, the presence of one or more vitamins such for example as biotin may be desirable to maintain maximum growth rate.

It is also desirable to add a non-toxic antifoaming agent to the substrate medium to control foaming during the fermentation.

The substance produced according to the present invention may be isolated in any suitable manner known in the art. Thus the resultant mycelium may be recovered by separation, washing, filtration and drying. In this connection, however, it has been found that if the moisture content of the substance is reduced below a critical level of about 50 percent (w/w) by filtration under pressure the subsequent drying methods employed are not subjected to such stringent temperature limitations which is an important factor in the economic processing of these materials. The method of drying must not cause damage to the nutritional value of the mycelium and may be drying in a current of air at C. or freeze dryingf The fungal mycelium produced by the process of the present invention shows very good water binding capacity and maybe useful as a thickening and gelling agent. Not being an isolate, it retains its vitamins as well as other nutritionally available materials such as lipids and some carbohydrates. Fungal mycelium has satisfactory baking characteristics which are of value in protein enriched breads, breakfast foods and food snacks. The fungal mycelium, because of its filamentous structure, can be baked, fried or puffed into a rice-like article and presented to many communities as a food comparable in appearance and acceptability with conventional foods which they are accustomed to eating.

Following is a description by way of example of methods of carrying the invention into effect.

EXAMPLE 1 l0 Liters of the following culture medium were prepared sterilised in a stirred fermenter vessel.

Cane molasses to provide 6% w/v sugar Ammonium sulphate Potassium dihydrogen phosphate Calcium carbonate 1.0% (to control pH,

otherwise gaseous ammonia could be used on automatic control) Tap water Antifoam: Polypropylene glycol 2000 2 ml. was

added to prevent foam formation The fermenter incubated at 30C. was then stirred at 800 rpm with a disc turbine and 1 VVM of sterile air. passed through. In 30 to 40 hours, the grown mycelium 1 5 was removed from the fermenter, centrifuged, washed with water and dried in a current of air at 75C."

EXAMPLE 2 This is an example of a starch preparation using potatoes as the starch source.

Washed potatoes were placed with water in a jacketed stirred vessel, brought to boiling with stirring and boiled until the whole was a uniform mash. The temperature was then reduced to 65C., pH adjusted to' 6.0-6.5 and heat stable bacterial a-amylase added and the stirring continued. After 20 minutes the liquified material was passed through a sieve in order to remove the potato skins, which are not attacked by the enzyme. This solubilised solution was then adjusted with water to give a solution that contained 6 percent of sugars.

400 l. of the following culture medium were prepared and sterilised in a stirred fermenter vessel:

Potato (treated as described above to provide) 6.0% w/v. sugar Ammonium sulphate 0.25%

KH PO. 0.3%

Tap water Antifoam soyabean oil (sufficient to prevent excessive foaming) Total Nitrogen (Kjeldahl) Amino Nitrogen Analysis Sulphur amino acids represent 3.40 percent of the total amino acids.

EXAMPLE 3 400 l. of the following culture medium were prepared and sterilised in a stirred fermenter vessel.

Cane molasses to provide 6.0% w/v sucrose Ammonium sulphate 0.25%

KH PO 0.20%

Corn steep liquor (50% TS) 1.0%

Antifoam Polypropylene glycol 2000 (sufficient to prevent excessive foaming) Total Nitrogen (Kjeldahl) 0: Amino Nitrogen Analysis Sulphur amino acids represent 2.37 percent of the total amino acids. r

EXAMPLE4 400 l. of the following culture medium were prepared and sterilised in a stirred fermenter vessel.

Spray dried milk whey (7% w/v) (to provide) 6.0% w/v lactose Corn steep liquor (50% TS) 0.4%

Ammonium sulphate 0.4%

KH PO, 0.2%

Tap water Antifoam Polypropylene glycol 2000 (sufficient to prevent ex cessive foaming) pH 5.5 (no control required). The process and other conditions were as described in Example 2. At approximately 72 hours mycelium was recovered as in Example 2.

Analysis Total Nitrogen (Kjeldahl) 0: Amino nitrogen Sulphur amino acids represent 4.31 percent of the total amino acid.

8 l. of the following culture medium were prepared and sterilised and added to a sterile fermenter.

Glucose 2.80% w/v Corn steep liquor (50% TS) 0.50% v/v KH PO, 0.20% w/v (NH) SO. 0.25% w/v Antifoam Polypropylene glycol 2000 (sufficient to prevent excessive foaming) pH maintained at 5.0 with gaseous ammonia. The seed inoculum was 10 percent of our strain of Penicillium notatum chrysogenum 1.113 (1M1 142385). The fermenter temperature was controlled at 30C. The fermenter was stirred at speeds of up to 800 rpm. with slow increase of the stirrer speed during the course of the batch to maintain the level of dissolved oxygen in the culture at a non-growth limiting concentration. The impeller was a single disc-turbine impeller of 11.5 cm. diamter. Aeration was at l VVM. After 25.5 hours a sample of the mycelium gave, on a dry weight basis a total Nitrogen of 7.51 percent.

EXAMPLE 6 The procedure of Example 5 was repeated with the exception that the culture used was our strain of Penicillium notatum chrysogenum 1.195 )IMI 142386) and the pH was controlled at 6.0. After 20.5 hours a sample of the mycelium gave, on a dry weight basis, a total Nitrogen of 7.53 percent.

Our strain of Penicillium notatum chrysogenum Cl and the variants thereof may be replaced in the above exampless by other strains of Penicillium notatum and Penicillium chrysogenum and Penicillium notatum chrysogenum that are non-toxic and contain no other undesirable metabolites such for example as the antibiotic penicillin.

a-amino nitrogen, characterized by being penicillin free during its growth and containing a non-toxic strain of Penicillium notatum or Penicillium chrysogenum. 

1. A NONVIABLE PENICILLIN FREE FUNGAL MYCELIUM FEMENTATION PRODUCT POSSESSING A HIGH NET PROTEIN UTILIZATION VALUE ON RAT ASSAYS OF AT LEAST70 BASED ON THE A-AMINO NITROGEN, CHARACTERIZED BY BEING PENICILLIN FREE DURING ITS GROWTH AND CONTAINING A NONTOXIC STRAIN OF PENICILLIN NOTATUM O PENICILLIN CHRYSOGENUM. 